ABSTRACT

Context Increasing evidence demonstrates that
atherosclerosis is an immunologically mediated disease in which the
secretion of atherogenic and atheroprotective cytokines, by
infiltrating blood mononuclear cells, plays an important role. It is
not known whether long-term exercise alters this atherogenic and
atheroprotective activity directly.

Objective To determine the effect of long-term exercise on the
atherogenic activity of blood mononuclear cells in persons at risk of
developing ischemic heart disease.

Design Before-after trial using a 6-month individualized,
supervised exercise program, with an enrollment period from December
1996 to October 1997.

Main Outcome Measures Blood levels were compared at
baseline and after the exercise program had been completed for the
following: spontaneous and phytohemagglutinin-induced production of
interleukin 1 α, tumor necrosis factor α, and interferon gamma
(atherogenic cytokines), and interleukin 4, interleukin 10, and
transforming growth factor beta 1 (atheroprotective cytokines) by blood
mononuclear cells; lymphocyte phenotypes and mitogenic responses to
phytohemagglutinin; and serum C-reactive protein levels.

Results Subjects exercised for a mean of 2.5 (range,
0.3-7.4) hours per week. Mononuclear cell production of atherogenic
cytokines fell by 58.3% (P<.001) following the exercise
program, whereas the production of atheroprotective cytokines rose by
35.9% (P<.001). Changes in transforming growth factor beta
1 and in phytohemagglutinin-induced atherogenic cytokine production
after the exercise program were proportionate to the time subjects
spent performing repetitive lower-body motion exercises
(P<.02), indicating a dose-response relationship. After the
exercise program, changes in cellular function were reflected
systemically by a 35% decrease in serum levels of C-reactive protein
(P=.12).

Figures in this Article

Although the age-adjusted death rate due to cardiovascular disease (CVD) has
declined in the past 25 years, heart disease remains the leading cause
of death in the United States, accounting for 733,834 deaths, or
31.6% of total mortality, in 1996.1

A number of studies have shown that moderate-intensity physical
activity reduces the incidence of all-cause mortality, particularly
deaths due to CVD.2- 9 The accumulated evidence on the
health benefits of physical activity prompted participants in a
National Institutes of Health Consensus Conference to recommend that
"children and adults alike should set a goal of accumulating at least
30 minutes of moderate-intensity physical activity on most, and
preferably all, days of the week."10

Despite the documented health benefits, the mechanism whereby
physical activity prevents CVD is incompletely understood, although it
is probably multifactoral. Risk factors such as
hypertension,11 obesity,12
hyperlipidemia,13 and insulin resistance14 may
respond favorably to moderate levels of physical activity, thereby
protecting against CVD. However, because exercise also protects against
CVD in smokers and in persons
without evident risk factors,6 it
appears to favorably influence the course of atherogenesis in ways yet
to be discovered. Whatever the reasons, reports have documented a
strong and independent association of low cardiorespiratory fitness and
low levels of physical activity and the risk of death due to
CVD.2- 10

There is increasing evidence that atherosclerosis may be an
immunologically mediated disease.15- 24 Early atherosclerotic lesions contain activated immune cells, including
CD4+ and CD8+ T cells, monocytes, macrophages,
and endothelial cells. These cells are responsible for the local
production of a variety of cytokines that have been identified in
atherosclerotic lesions, including interleukin (IL) 1, IL-2, IL-4,
IL-6, IL-10, tumor necrosis factor alpha (TNF-α), interferon gamma
(IFN-γ), and transforming growth factor beta (TGF-β). There is
provocative but, as yet, inconclusive evidence that this immune
reaction may be in response to an infectious agent,25- 27
heat shock protein (HSP) 60,19,23 or oxidized low-density
lipoprotein (LDL).28 Whatever the inciting events, in
transplanted human hearts, activation of coronary endothelial cells is
predictive of the subsequent development of coronary
atherosclerosis,29 lending credence to the importance of the immune response in the early stages of CVD. Indirect evidence is
also found in reports documenting an association between blood levels
of acute-phase reactants, notably C-reactive protein
(CRP),30,31 fibrinogen,31 and C332
and the future risk of heart attack. Acute phase reactants are produced
by hepatocytes in response to several cytokines, notably IL-1, IL-6,
and TNF-α, and often serve to minimize tissue damage that follows an
inflammatory response.

In this study, we investigated the possibility that long-term
exercise benefits persons at risk of developing ischemic heart disease
by favorably altering the production of cytokines with atherogenic and
atheroprotective properties. According to current evidence, the
cytokines with atherogenic properties include IL-1, TNF-α, and
IFN-γ.16- 24 The cytokines with atheroprotective
properties include IL-4, IL-10, and TGF-β.33- 50 Because
blood leukocytes provide the major source of immune cells in
atherosclerotic lesions21 and because it is not feasible to obtain these cells from arterial biopsy specimens, we have chosen to
measure the effect of exercise on the production of these cytokines by
blood mononuclear cells. We have also determined the effect of exercise
on serum levels of CRP, an acute-phase protein that has been speculated
to serve as a marker of the severity of the immune-mediated
inflammatory response in atherosclerosis.30

METHODS

Subjects

This study was approved by the institutional review board of East
Tennessee State University, Johnson City. Each participant read and
signed the informed consent in the presence of an investigator.

Subjects were recruited from the general public by placing an
outline of the study and a request for volunteers in 3 local
newspapers. A total of 110 persons responded and were screened for
participation in the study. Of these, 54 had serum C3 and/or CRP levels
that placed them at greater risk of future heart attack (risk ratio
≥1.7),30,32 and they underwent exercise treadmill testing
(ETT) using a modified Bruce protocol. Fifty-two subjects had normal
ETT results and qualified for final inclusion in the study. These
subjects were enrolled in a hospital-based wellness center where, after
analysis of their medical history and previous levels of activity, they
were assigned to supervised and individually tailored exercise
programs. To minimize seasonal influences on the results, 4 to 5
subjects were enrolled each month over an 11-month period extending
from December 1996 to October 1997. Subjects were not paid for
participating in the study. Forty-three subjects (18 men, 25 women)
successfully completed the study. Nine subjects withdrew for personal
reasons. Summaries of subject demographics and risk factors for CVD are
shown in Table 1.

Immunologic studies were performed at baseline and after completion of
the 6-month exercise program. All blood samples were drawn in the
morning at approximately the same time of day.
Subjects were instructed not to exercise for at least 24 hours prior to
blood drawing.

Blood CRP

C-reactive protein serum levels were assayed before and after the
exercise program by radial immunodiffusion using CRP LL and CRP EL
Nanorid kits (The Binding Site Ltd, Birmingham, England).

Mitogenic Assays

Mitogenic responses were measured by adding
methyl-3H-thymidine (0.74 MBq, 50 µL) to culture samples
removed 6 hours prior to supernatant harvesting. Labeled samples were
incubated under 5% carbon dioxide at 37°C for an additional 6 hours
and the cells collected on glass fiber filter paper using a Mash II
Cell Harvester (Microbiology Associates, Walkersville, Md). Samples
were air dried, placed in vials containing Scinti-Verse II, and assayed
with a Beckman LS 9800 liquid scintillation counter (Beckman,
Fullerton, Calif). Proliferative responses are expressed as net
counts per minute (Δ cpm), calculated as cpm in PHA-stimulated
cultures minus cpm in unstimulated cultures. The mitogenic assay used
in this study had been optimized in accordance with standard procedures
using varying concentrations of PHA, cells per well, and incubation
times.51

Statistical Analysis

Statistical analysis was performed using STATISTICA 5.1 F
(Statsoft, Inc, Tulsa, Okla). The 2-sided t test for paired
samples was used to determine the significance of differences between
cytokine, lymphocyte phenotype, and CRP measurements in blood samples
taken before and after the exercise program. A 2-sided t test
for independent samples was used to determine within-group differences.
A χ2 analysis was used to determine the significance of
differences between expected and observed frequencies. Unless stated
otherwise, results are expressed as the mean (SEM).

RESULTS

Production of
Atherogenic Cytokines

The production of IFN-γ and TNF-α by blood mononuclear cells was
significantly attenuated in both PHA-negative and PHA-positive cultures
by exercise (Table 2). Compared with
baseline values, IFN-γ levels fell by 44% and 71% and TNF-α
values by 28% and 51% in cultures with and without added PHA,
respectively (P≤.007). In addition, IL-1α secretion fell by
13% in PHA-stimulated cultures (P=.047).
Overall the production of atherogenic cytokines fell by 58.3% after
the exercise program (P<.001).

Production of
Atheroprotective Cytokines

In contrast to the effects of exercise on the production of atherogenic
cytokines, the production of IL-4, IL-10, and TGF-β1 by blood
mononuclear cells was significantly augmented by exercise (Table 2 In PHA-negative cultures, IL-10 levels increased by 940% and TGF-β1 by
43% (P≤.003). In PHA-positive cultures, significant changes
were seen in IL-4 (94% increase) and
TGF-β1 (37% increase) (P≤.001).
Overall the production of atheroprotective cytokines increased by
35.9% after the exercise program (P<.001).

Lymphocyte Phenotypes

There were no significant changes in the numbers or percentages
of lymphocyte phenotypes following exercise (Table 3).

Mitogenic Responses

Proliferative responses of lymphocytes to PHA decreased following
exercise, from a mean (SEM) value of 9142 (760) Δcpm to 3155 (584)
Δcpm (P<.001).

CRP Levels

C-reactive protein levels measured before the exercise program ranged
from 0 to 0.9 mg/dL in the lower quartile to 5.8 to 37.5 mg/dL in the
upper quartile, with a mean (SEM) value of 4.81 (1.09) mg/dL. Values
taken after the exercise program decreased by 35% to 3.13 (0.64) mg/dL
(P=.12) (2-sided t test). The
frequency of values in the upper quartile dropped by 50% following
exercise (P=.01) (χ2 analysis).

Exercise Parameters

The mean number of hours per week that subjects underwent
supervised exercise was 2.5 (range, 0.3-7.4 hours per week). The mean
duration of each exercise session was 70 minutes (range, 36-123
minutes), and the mean number of visits per week was 2 (range, 0.3-5.3
visits per week). The mean percentage of time spent doing different
exercises was as follows: weight lifting (35%), walking or running on
a treadmill (32%), cycling (16%), stretching (8%), aerobics (3%),
rowing (3%), climbing (2%), and skiing (1%).

Four subjects (9.3%) changed their diet to one that was lower in
energy intake and animal fat, and 2 of the 5 tobacco users discontinued
smoking during the study. By completion of the study, 15 subjects
(34.9%) had lost weight, with the mean weight for all subjects falling
from 80.1 to 77.4 kg (P=.02). There were no
changes in medications or alcohol consumption during the study.

Relationship of Exercise Parameters to Cytokine Production

The percentage decrease in atherogenic cytokine production
by PHA-stimulated mononuclear cells was proportionate to the time
subjects spent performing repetitive lower-body motion exercises (ie,
walking, running, cycling, rowing, climbing, aerobics, and skiing)
(r=−0.267, P=.002).
The correlation was most significant for IFN-γ
(P=.03) (Figure 1), followed by IL-1α
(P=.05) and TNF-α
(P=.09).

Figure. Effect of Exercise Duration on the Production of
Interferon Gamma (IFN-γ)
by Phytohemagglutinin-Stimulated
Mononuclear Cells

The average number of hours per week each subject spent doing
repetitive lower-body motion exercises (walking, running, cycling,
rowing, climbing, aerobics, and skiing) is plotted against the change
in IFN-γ production that occurred following these exercises
(r=0.3, P=.03). The
production of IFN-γ decreased in proportion to the duration of
exercise.

In contrast, both the spontaneous and PHA-induced production of
TGF-β1 increased in proportion to the time spent doing these
exercises (r≥0.354, P≤.02). No other correlations
were found between exercise parameters and main outcome measures.

Within-Group Variations

Blood mononuclear cells taken from men prior to exercise produced
more IFN-γ and TNF-α in cultures without added PHA than did
mononuclear cells taken from women (P≤.04). Following
exercise, men spontaneously produced more IL-1α and TNF-α than
women (P≤.004). In contrast, men had
lower circulating levels of CD4+ and
VLA-4+ T cells prior to and after exercise
(P≤.049), and lower levels of CD95+ T cells
following exercise (P=.03). Mitogenic
responses before the exercise program were also lower in men
(P=.03), whereas there was no difference in
proliferative responses to PHA in samples between men and women taken
after the exercise program.

Mononuclear cells of hypertensive subjects produced
significantly more IL-1α and TNF-α than those of normotensive
subjects following exercise (P≤.03). The production of
IL-1α and TNF-α was also higher in hypertensive subjects before the
exercise program, with differences approaching statistical significance
(P≤.13). This finding may explain in part the higher levels
of IL-1a and TNF-α in men, since they had a higher prevalence of
hypertension than did women.

No within-group differences could be demonstrated as a result of
menopause, estrogen therapy, use of aspirin or other medications,
alcohol consumption, obesity, hypercholesterolemia, smoking, diabetes
mellitus, dieting, weight loss, or smoking cessation during the study.

COMMENT

In our study, 6 months of moderate-intensity exercise attenuated blood
mononuclear cell production of cytokines with predominantly atherogenic
properties, while simultaneously augmenting the production of cytokines
with predominantly atheroprotective properties.

The decrease in atherogenic cytokine production by PHA-stimulated
mononuclear cells and the increase in mononuclear cell production of
the atheroprotective cytokine TGF-β1 after the exercise program were
proportionate to the time subjects spent in performing repetitive
lower-body motion exercises, indicating the existence of a
dose-response relationship between exercise and mononuclear cell
function in our subjects. The changes in immune cell function were
accompanied by a reduction in serum CRP, a possible systemic reflection
of decreased IL-1α and TNF-α production and a favorable sign in
persons at risk of developing CVD.30,31 Importantly, the
changes were unrelated to the ingestion of aspirin or other
medications, or to dieting, weight loss, or smoking cessation during
the study. Subjects included men and women who had been selected from
the general population and who had well-defined risk factors for
ischemic heart disease.

We found that exercise had a particularly significant attenuating
effect on the production of IFN-γ and TNF-α. Both of these
cytokines have been identified in early and advanced atherosclerotic
lesions and are postulated to play preeminent roles in
atherogenesis.16- 24 Tumor necrosis factor alpha is produced
primarily by monocytes and macrophages and is a potent proinflammatory
cytokine.33 Interferon gamma is produced in T-helper type 1 (Th1) lymphocytes, NK cells, and cytotoxic T lymphocytes and is the
most important cytokine regulating the activities of mononuclear
phagocytes35,52 and, therefore, cell-mediated immune
responses of the type seen in atherosclerotic lesions.16,17
Both cytokines can activate endothelial cells, monocytes, macrophages,
and smooth muscle cells, thereby contributing to leukocyte recruitment,
endothelial cell procoagulant activity, LDL oxidation, and foam cell
formation in atherosclerotic lesions.16- 24 The
importance of IFN-γ in atherogenesis has recently been demonstrated
in transgenic mice with targeted disruptions of the APOE gene
and the IFN-γ receptor gene (apoE 0/IFN-γR 0
mice).53 These mice demonstrate a substantial reduction in atherosclerotic lesion size, cellularity, and lipid accumulation,
and an increase in plasma concentrations of potentially
atheroprotective phospholipid/apoliprotein A-IV rich particles when
compared with APOE 0 mice, suggesting that IFN-γ promotes
and modifies atherosclerosis through its local effects in the arterial
wall as well as by its effects on plasma lipoproteins.

In contrast to the atherogenic cytokines, the production of
cytokines that possess predominantly atheroprotective properties was
augmented by exercise. In unstimulated blood mononuclear cell cultures,
the cytokine most affected was IL-10, followed by TGF-β1. The
secretory responses of PHA-stimulated cultures were generally similar,
except that IL-4 production was also up-regulated by exercise. These
cytokines effectively down-regulate delayed-type hypersensitivity
reactions of the type seen in atherosclerotic lesions, primarily by
suppressing mononuclear phagocyte and Th1 lymphocyte
function.33- 50,52 They inhibit endothelial cell, monocyte,
macrophage, and smooth muscle cell activation by down-regulating the
production of IL-1α and TNF-α by mononuclear phagocytes (IL-4,
IL-10, and TGF-β1), and IFN-γ production by Th1 lymphocytes (IL-10
and TGF-β). They also reduce IL-1 activity by augmenting the
production of IL-1 receptor antagonist.33 Although TGF-β
is thought to be primarily responsible for stimulating collagen and
proteoglycan synthesis by vascular smooth muscle cells in
atherosclerotic plaques, it inhibits the proliferation and migration of
these cells in atherosclerotic lesions48 and is thought to function primarily as an atheroprotective cytokine.44- 50 In
this regard, low plasma levels of TGF-β have been documented in
patients with ischemic heart disease,46 possibly due to its sequestration into an inactive pool by lipoproteins.45 It
is of note that, in our study, mononuclear cell production of TGF-β1
increased in proportion to time spent doing exercises traditionally
considered to be cardioprotective.

It is probable that the changes in blood mononuclear cell
function among the subjects after undergoing the exercise program
resulted, at least in part, from an augmented suppression of TNF-α
and IFN-γ production by the atheroprotective cytokines. Furthermore,
because blood leukocytes are thought to provide the main source of
immune cells in atherosclerosis, similar functional changes may have
occurred in mononuclear cells present in atherosclerotic
lesions.21 This possibility is supported indirectly by the
decrease in serum levels of CRP in our subjects after the exercise
program. It remains uncertain, however, what initiated these changes.
Since herpes viruses and chlamydia have been isolated from
atherosclerotic plaques,25- 27
it is possible that physical activity stimulated
the immune system to eradicate these agents. Alternatively, it may be
that exercise reduced levels of oxidized LDL or HSP 60 in fatty streaks
and atherosclerotic plaques in our subjects. Both of these molecules
have been postulated to be the target of an autoimmune attack in
atherosclerosis and are capable of independently activating endothelial
cells.19,23,28 Whatever the explanation, our finding that
moderate long-term exercise reduces blood mononuclear cell production
of cytokines with predominantly atherogenic properties, while
simultaneously increasing their production of cytokines with
predominantly atheroprotective properties, provides an insight as to
how physical activity helps protect against CVD.

Figure. Effect of Exercise Duration on the Production of
Interferon Gamma (IFN-γ)
by Phytohemagglutinin-Stimulated
Mononuclear Cells

The average number of hours per week each subject spent doing
repetitive lower-body motion exercises (walking, running, cycling,
rowing, climbing, aerobics, and skiing) is plotted against the change
in IFN-γ production that occurred following these exercises
(r=0.3, P=.03). The
production of IFN-γ decreased in proportion to the duration of
exercise.

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